3.1. Proximate chemical analysis of broad bean (Vicia faba L.) pods
The data in Table (1) showed the chemical composition of the broad bean pods in their green state after oven drying at 50o C. The chemical analysis revealed that the dried pods contain moisture (9.27%), protein (8.38%), fat (0.38%), ash (7.22%), fibers (14.59%) and carbohydrates (60.16%). Our findings indicated that the dried green pods have a high content of carbohydrates and dietary fiber, which differs from the results of Mateos-Aparicio et al. [38], who reported that the broad bean pods contain high contents of protein (13.6%), and dietary fiber (40.1%), fat (1.3%), and ash (6.3%) on a dry weight basis. As demonstrated by Mejri et al. [18], broad bean pods had a high moisture content (79.26% on a wet weight basis), while their content of proteins, carbohydrates, lipids, and dietary fiber was 13.81, 18.93, 0.92 and 57.46% (on a dry weight basis), respectively. Also, Vernaleo et al. [39] stated that fava beans are a rich source of dietary fiber and high in phytonutrients such as isoflavone and plant sterols. The differences in the chemical composition could be attributed to the geographical location, handling and processing or the variety of the broad bean.
Table (1): Proximate chemical composition of broad bean pods (g/100g DW):
components | Moisture | Protein | Fat | Ash | Fibers | Carbohydrates |
| 9.27 ± 0.08 | 8.38 ± 0.06 | 0.38 ± 0.03 | 7.22 ± 0.10 | 14.59 ± 0.03 | 60.16 ± 0.16 |
Each value is the mean ± SD |
3.2. Mineral contents of broad bean pods (mg/100g DW)
Results in Table (2) revealed that the dried broad bean pods contain 937.2, 32.09, 340.4, 2.189, 3483, 4.442 and 340.1 mg/100g of calcium (Ca), iron (Fe), magnesium (Mg), manganese (Mn), potassium (K), copper (Cu) and phosphorus (P), respectively. It is evident that the broad bean pods are a good source of K, Ca, Mg and Fe. Results are in the same line with Vernaleo et al. [39], who stated that fava beans are a good source of phosphorus, iron, copper, manganese, calcium, magnesium, and potassium. Mateos et al. [38] found that the broad bean pods (byproducts) contain the most important minerals; potassium, calcium, and iron.
Table (2): mineral contents of broad bean pods (mg/100g DW):
ة Mineral | Concentration (mg/100g) |
Ca | 937.2 |
Fe | 32.09 |
Mg | 340.4 |
Mn | 2.189 |
K | 3483 |
Cu | 4.442 |
P | 340.1 |
3.3. Phytochemical screening of broad bean pods methanolic extract
As shown in Table (3) the phytochemical screening revealed that the methanolic extract of broad bean pods contain phenolic compounds, flavonoids, glycosides, tannins, alkaloids and saponins.
All phytochemicals were found in the ethanolic extract of Vicia faba L., with the exception of anthracenosides, sterols, and triterpenes (Fabaceae). The aqueous extract included fewer phytochemicals like tannins, alkaloids, glycosides, sterol, triterpenes, and saponins than the ethanolic extract. Additionally, reducing sugars were found in the ethanolic extract exclusively [40]. Faba beans contain polyphenols in a variety of plant components, including the leaves, roots, and seeds [41]. The contents of cotyledons from faba beans are higher than those of their hulls or complete seeds. According to recent studies, faba beans and their derivatives may be an appropriate meal for the treatment of hypertension, diabetes, and cardiovascular disease [42].
Table (3): Phytochemical screening of broad bean pods methanolic extract:
Glycosides | Phenols | Tannins | Alkaloids | Flavonoids | Saponins |
+++ | +++ | + | + | ++ | + |
3.4. Total phenols and total flavonoids of the broad bean pods methanolic extract
The total phenols and flavonoids of the methanolic extract of broad bean pods are represented in Table (4). Data showed that the phenol content was 286 mg GAE/g, and the total flavonoid content was 105mg QE/g. Our results are higher than that of Mejri et al. [18], who reported that the total phenolic compounds (TPC) in the methanol extract of broad bean pods were 115.21 mg GAE per g extract, while the total flavonoids compounds (TFC) were 47.34 mg QE per g extract. Valente et al. [43] found that the total free phenols in the dried pods ranged from 10.87 to 26.34 mg/100g in different varieties of faba bean pods, while the total esterified phenolics ranged from 8.76 to 26.72 mg/100g dry weight. According to Chan et al. [44], the methanolic extract of broad bean pods included a high concentration of total phenolics and flavonoids, including numerous polar aglycones and flavonoid glycosides. The literature needs to characterize the phenolic content sufficiently. According to one study on thirteen genotypes of broad bean pods grown in the same area and subjected to the same conditions, TPC ranged from 56.97 to 149.21 mg EGA per g. The genotypic effect of TFC in the same study had values ranging from 10.23 to 45.92 mg RE per g [45].
Table (4): Total phenols and flavonoids of broad bean pods methanolic extract
Total phenols | (286 mg GAE / g) |
Total flavonoids | 105 mg QE/g |
3.4. Antioxidant activity of broad bean pods methanolic extract
3.4.1. DPPH assay.
The antioxidant activity of the broad bean pods' extract was measured by 1,1-diphenyl-2-picryl hydrazyl (DPPH) assay. The results in Table (6) and Fig. (1) revealed that the DPPH scavenging percentage increased by increasing the pods' extract concentration. The highest value of DPPH scavenging activity which reached 80.5%, was achieved by the extract concentrations of 1000 ug/ml. The concentrations of 500 and 250 ug/ml of the pod extract also showed high levels of DPPH scavenging activity, reaching 73.7 and 65.7%, respectively. The IC50 is a parameter for measuring the concentration of the antioxidant substance needed to decrease the initial DPPH concentration by 50%. Therefore, the low levels of IC50 indicate high antioxidant activity. The obtained results showed that the IC50 concentration of 87.35 ug/ml is small, which means that the broad bean pods have high antioxidant activity. Our results agree with Mateos-Aparicio et al. [19], who reported that the polyphenols extracted from broad bean pods exhibited high reducing power and free-radical scavenging activity. The antioxidant activity of the broad bean pods is attributable to their high content of phenolic compounds [46]. It is well known that most plants contain natural products which play essential roles in fighting diseases including cancer. The broad bean pods are rich in fiber, phenolic acids, and flavonoids, which can prevent the oxidation of cell membranes and protect the cells from free radicals and toxic substances. It has also been found that the tannins in faba beans could provide hydroxyl radical scavenging activity [47]. It was hypothesized that the extract of the pods prevents the reaction of hydroxyl radicals with the hydrogen atoms of the sugar moiety of DNA and hence protects DNA from damage [48].
Table (6): Antioxidant activity of broad bean pods methanolic extract by using DPPH assay.
Extract Conc. (µg/ml) | OD | DPPH scavenging % |
1000 | 0.295 | 80.5 |
500 | 0.397 | 73.7 |
250 | 0.518 | 65.7 |
125 | 0.687 | 54.5 |
62.50 | 0.879 | 41.8 |
31.25 | 0.978 | 35.3 |
15.625 | 1.158 | 23.4 |
7.8125 | 1.224 | 19.0 |
3.90 | 1.305 | 13.6 |
1.95 | 1.356 | 10.3 |
IC50 (87.35 µg/ml) |
3.4.2. Antioxidant activity of broad bean pods methanolic extract using reducing power
Table (5) shows that the reducing power of broad bean pods' methanolic extract increased by increasing the concentration. It varied from 33.9 to 79.1% by the extract concentrations of 100, 200, and 400 mg/ml. The IC50 values reached 177.32 mg/ml.
Any substance with a reducing power will combine with potassium ferricyanide (Fe3+) to generate potassium ferrocyanide (Fe2+), which will then react with ferric chloride to form ferric- ferrous complex, or pearl's Prussian blue, which is absorbed at 700 nm [49]. Reductive action and antioxidant activity are connected [50]. As mentioned before, the BBP methanol extract demonstrated potent antioxidant activity (60.72%). It was also shown that the lowest IC50 for the DPPH and ABTS assays is accompanied by the compound's highest free radical scavenging activity [18].
Table (5): Antioxidant activity of broad bean pods methanolic extract using reducing power
Concentration mg/ml | 100 | 200 | 400 |
Inhibition % | 33.9 | 61.04 | 79.1 |
IC50 (177.32mg/ml) | |
3.5. Anti-inflammatory activity of broad bean pods methanolic extract
HRBC (human red blood cells) method was used for the in vitro study the anti-inflammatory effect of the broad bean pod extract. The erythrocyte membrane and the lysosomal membrane are comparable; therefore, by stabilizing the erythrocyte membrane, the extract from broad bean pods may stabilize the lysosomal membrane.
The data in Table (7) showed that all the extract concentrations exhibited a significant reduction in RBCs hemolysis, where the maximum inhibition percentage reached 66.7% with a concentration of 1000 ug/ml. However, the lowest concentration (400 ug/ml) of pod extract showed an inhibition percentage of about 50.8%. It is clear from these results that the pods' extract of broad beans has anti-inflammatory activity in the studied models.
The hypotonic solution causes hemolysis of RBCs because of fluid accumulation in the cells, which leads to rupturing of their membranes. The injury occurred in the RBCs makes them more susceptible to lipid oxidation via the free radicals causing the moving of some components, such as protein and fluids, to the tissues, which are similar to that produced during inflammation [51].
The extract of broad bean pods prevents the oxidation of RBCs membranes lipids and preserves them. In addition, it stabilizes the RBCs membrane by preventing the production of lytic enzymes and active inflammatory mediators.
Our results revealed that the pods of broad bean extract contain flavonoids, alkaloids, and saponin, to which the anti-inflammatory is attributed. Many studies showed that the plants' flavonoids have antioxidant and anti-inflammatory effects [52, 53, 54]
Their anti-inflammatory effect may be due to their ability to inhibit the enzymes which contribute to the production of inflammatory mediators and the enzymes of arachidonic acid metabolism [55, 56]
Table (7): Anti-inflammatory activity of broad bean pods methanolic extract
Conc. (ug/ml) | Hypotonic Ab. Mean | Sample with Isotonic solution Ab. | Hemolysis Inhibition % |
Control | 0.759 | | 0 |
1000 | 0.291 | 0.057 | 66.7 |
800 | 0.341 | 0.051 | 59.0 |
600 | 0.367 | 0.045 | 54.9 |
400 | 0.394 | 0.041 | 50.8 |
200 | 0.429 | 0.039 | 45.8 |
100 | 0.503 | 0.036 | 35.4 |
3.6. Antimicrobial activity of broad bean pods methanolic extract:
The antimicrobial activity of the methanolic extract isolated from broad bean pods was assessed in vitro by the agar well diffusion method against four pathogenic bacteria strains and two kinds of fungi. The bacteria strains included two- gram-positive (Bacillus subtilis and Staphylococcus aureus) and two gram-negative (Escherichia coli and Pseudomonas aeruginosa), while the two fungi were Candida albicans and Aspergillus fumigatus.
Table (8) results revealed that the pods' extract prevented the growth of B. Subtilis, Staph. aureus and E. Coli in addition to the two fungi, Pseudomonas aeruginosa and Candida albicans. Their corresponding inhibition zones were 16, 17, 15, 28 and 23 mm, respectively. On the other hand, the Asp. fumigatus was resistant to the pod extract, which showed no activity in this respect. It was also noticed that the antimicrobial effect of the pod extract was more effective than the gentamycin (reference control) against Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. These results agreed to a less extent with those of Peyvast and Khorsandi [57], who stated that the ethanolic extract of the seed hull of broad bean exhibited antimicrobial activity against E. Coli, B. subtilis and staph. aureus.
Table (8): Antimicrobial activity of broad bean pods methanolic extract against some microorganisms
Pathogenic microorganism | Diameter in mm |
Sample | Control |
Bacillus Subtilis (ATCC 6633) | 16 | 25 |
Staph.aureus (ATCC 6538) | 17 | 15 |
Escherichia coli (ATCC 8739) | 15 | 17 |
Pseudomonas aeruginosa (ATCC 90274) | 28 | 22 |
Candida albicans (ATCC 10221) | 23 | 21 |
Aspergillus Fumigatus | NA | 15 |
*Antimicrobial activity was determined by using agar diffusion, Disc diameter: 6.0 mm (100 µl was tested).
* NA: No activity
* All samples were dissolved in normal saline (0.9% NaCl).
* Saline didn’t have antimicrobial activity against all tested pathogenic strains
* Inhibition zones were represented as mm.
* Control: Gentamycin
3.7. Anticancer activity of broad bean pods methanolic extract:
Liver cancer is the world's fourth most common cause of death [58]. It is the most cause of mortality and morbidity-concerning cancer because of the widespread hepatitis C in the last decades. HCV infection is the leading cause of cirrhosis [59] which is one of the risk factors for liver cancer [60]. Because of the side effects of the drugs used in cancer treatment, we searched for natural products as novel anticancer remedies.
In this study, the effect of methanolic extract of broad bean pods on human hepatocellular carcinoma (HepG2) and prostate cancer (PC3) cells was investigated. The results in Table (9) and figures (2&3) showed that the methanolic extract of broad bean pods decreased the cell viability and increased cell toxicity of both HepG2 and PC3 in a concentration-dependent manner. It was noticed that the low extract concentration of 31.25 has no significant effect on the two mentioned cells' viability. On the other hand, all the other concentrations caused significant decreases in the viability of the two kinds of cells, increasing cell toxicity. The viability percentage of the doses 125, 250, 500 and 1000 ug/ml was 49. 54%, 35.52%, 18.46% and 4.07%, respectively in HepG2 cells, while it was 48.63% 21.32%, 11.59% and 3.43% in PC3 Cells, respectively. The methanolic extract of the pods under study exhibited high toxicity to the HepG2 and PC3 cells where the toxicity percentage reached more than 96% with a concentration of 1000 ug/ml. The IC50 values of the pod extract were observed at concentrations of 126.97 µg/ml for HepG2 cells and 125.12 µg/ml for PC3 cells, which are good results as an anticancer agent.
Morphological analysis:
As seen in Figs. 2 and 3, the methanolic extract of broad bean pods caused remarkable alterations in the shape of the cells compared to the control. Furthermore, the changes in the cell’s morphology increased with the increase of extract concentration, where a large amount of dead and detached cells indicate the toxic effect of the pods extract on the proliferation of the tumor cells after 24h incubation of the HpeG2 and PC3 Cells with the methanolic extract of the broad bean pods. It was observed that the low concentration of 31.25 ug/ml did not cause any significant alterations. In contrast, the higher concentrations caused substantial changes in the morphology of the tumor cells, which increased by increasing the extract concentration.
Many studies focused on benefiting from the plant byproducts that burden the environment. For example, some fruits and vegetable wastes can be used as feed for cattle and sheep; others can be used in fertilization [61]. Recent studies tried to benefit from the bioactive components found in plant and vegetable wastes as nutraceuticals used for human health as antioxidants and anticancer [62]. Other studies reported that the legumes' polyphenols and micronutrients have important biological values [63, 64]. The role of polyphenols in protecting the human body from chronic diseases such as cardiovascular diseases, diabetes, asthma, cancer, and inflammation was assessed [65]. The anticancer activity of the broad bean pod extract is attributed to its content of phenolic compounds.
P-coumaric and ferulic acids which are among the phenolic acids found in the pods of the broad beans are known for their anticancer activity against different types of cell lines [66].
In a recent study, Ceramella et al. [67] showed that three extracts of broad bean pods (acetone, methanol and ethanol 70%) have excellent antioxidant activity via studying DPPH and ABTS assays. They also found that the three extracts exhibited an enjoyable anticancer activity against melanoma SK- Mel-28 cells.
Table (9): Effect of broad bean pods methanolic extract against liver and prostate cancer in vitro
Pods extrac Conc. (µg/ml) | Liver (HepG2) | Prostate (PC3) |
Viability | Toxicity | Viability | Toxicity |
Control | 100.0 a | 0.00f | 100.00a | 0.00f |
1000 | 4.07f ± 0.12 | 96.02a ± 0.26 | 3.43f ± 0.78 | 96.44a ± 0.59 |
500 | 18.46e ± 2.79 | 81.54b ± 2.79 | 11.59e ± 0.98 | 88.41b ± 0.98 |
250 | 35.52d ± 7.21 | 64.48c ± 7.21 | 21.32d ± 2.83 | 78.68c ± 2.83 |
125 | 49.54c ± 2.81 | 50.40d ± 2.91 | 48.63c ± 3.58 | 51.38d ± 3.58 |
62.5 | 89.99b ± 1.49 | 10.01e ± 1.49 | 89.52b ± 2.62 | 10.48e ± 2.62 |
31.25 | 99.83a ± 11.90 | 0.17f ± 11.90 | 99.17a ± 2.52 | 0.83f ± 2.52 |
IC50 dil. | 126.97 µg/ml | 125.12 µg/ml |
Many studies of plant extracts revealed that polyphenols could play a crucial role in preventing and progressing chronic illnesses related to inflammation, such as cardiovascular diseases, obesity, neurodegeneration, cancers, and diabetes, among other conditions [68, 69]. Polyphenols can suppress toll-like receptor (TLR) and pro-inflammatory genes’ expression. The antioxidant activity of polyphenols is attributed to their ability to inhibit enzymes that contribute to the production of eicosanoids and their anti-inflammation properties. For example, they inhibit certain enzymes producing reactive oxygen species ROS such as xanthine oxidase and NADPH oxidase (NOX). At the same time, they increase other endogenous antioxidant enzymes like superoxide dismutase (SOD), catalase, and glutathione (GSH) peroxidase (Px). On the other hand, they inhibit phospholipase A2 (PLA2), cyclooxygenase (COX) and lipoxygenase (LOX), causing reductions in the production of prostaglandins (PGs) and leukotrienes (LTs) and inflammation antagonism. These effects on the immune system led to health benefits for different chronic inflammatory diseases [69].